Method for extracting kapur through compound bacterium fermentation

Abstract

The invention provides a method for extracting kapur through compound bacterium fermentation, which comprises the following steps: drying leaves of kapur burmannii chvar.Borneol by microwaves, mixing and crushing to obtain leaf powder, adding a compound enzyme of xylanase, hemicellulase, lipase and pectinase for enzymolysis, taking a citric acid-sodium citrate buffer solution as a solvent, carrying out enzymolysis, heating for inactivation, filtering, washing, and drying to obtain a finished product. The borneol type cinnamomum burmannii leaves are obtained by fermenting the borneol type cinnamomum burmannii leaves with a complex microbial inoculant, extracting a fermentation product with an ionic liquid-ethanol mixed solution, extracting with petroleum ether, condensing and refluxing with absolute ethyl alcohol and performing vacuum drying to remove an organic solvent, the yield of the borneol type cinnamomum burmannii leaves is high, release of IFN-beta and IL-6 inflammatory mediators can be remarkably reduced, inflammatory response is relieved, and the borneol type cinnamomum burmannii leaves are prepared. The inflammation process is inhibited, the effect of treating or preventing chronic pharyngitis is achieved, and the effects of improving sleep and relieving emotion are achieved.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation, and in particular, to a method for extracting borneol by composite bacteria fermentation. Background Art

[0002] Borneol is a natural monoterpene compound, which is widely used in traditional Chinese medicine and modern pharmaceutical preparations due to its anti-inflammatory, antibacterial, analgesic and sedative pharmacological activities. At present, the main sources of borneol include synthetic preparation and extraction from plants. However, traditional extraction techniques (such as steam distillation and organic solvent extraction) have many deficiencies, such as low extraction efficiency, easy damage to the thermosensitive components of borneol, and possible problems such as solvent residues. Therefore, developing an efficient, green and safe method for extracting borneol is of great significance for promoting its medicinal development.

[0003] In recent years, the composite enzymatic hydrolysis technology has been widely used in the extraction of plant active ingredients. By utilizing the synergistic effect of multiple enzymes (such as xylanase, hemicellulase, pectinase and lipase), it can significantly destroy the plant cell wall structure, promote the release of active ingredients, and improve the extraction efficiency. At the same time, microwave drying technology, due to its characteristics of rapid, efficient and uniform heating, can effectively remove moisture while retaining plant active ingredients, and improve the stability in the powder preparation process. However, the combined application of composite enzymatic hydrolysis and microwave drying technology in the extraction of borneol is not sufficient, and its potential needs to be further explored.

[0004] Fermentation, as a means of biotransformation, has been proven to be able to significantly improve the bioavailability and pharmacological activity of plant active ingredients. Especially composite bacteria fermentation, through the metabolic activities of microorganisms, can further release the active ingredients in plant raw materials and improve the extraction yield. In addition, green solvent technology (such as ionic liquids) has received extensive attention in the extraction of natural products in recent years due to its low toxicity, environmental friendliness and selective extraction ability for target products. However, how to combine fermentation technology with green solvents for the extraction of borneol to further improve the extraction efficiency and retain its medicinal activity is a technical problem that has not been fully solved in the related fields at present. Summary of the Invention

[0005] In order to solve the above problems, the present invention first provides a method for extracting borneol by composite bacteria fermentation. This method combines multiple technologies such as microwave drying, composite enzyme hydrolysis, composite bacteria fermentation and green solvent extraction, significantly improves the extraction efficiency of essential oil in Cinnamomum burmanni Blume var. macrophyllum leaf, and at the same time retains its active ingredients, and has the effects of anti-inflammatory, reducing inflammatory response and treating chronic pharyngitis.

[0006] In some embodiments, the method for extracting borneol by composite bacteria fermentation provided by the present invention comprises the following steps: S1 Pretreatment: The leaves of Cinnamomum burmannii chvar. Borneol are mixed and ground after microwave drying to obtain leaf powder. S2 Enzymolysis: The solid powder obtained in step S1 is added to a citric acid - sodium citrate buffer solution, and a complex enzyme composed of xylanase, hemicellulase, lipase and pectinase is added for enzymolysis. After enzymolysis, the enzyme is inactivated by heating, filtered under reduced pressure, and vacuum dried to obtain an enzymolysis product. S3 Fermentation: The enzymolysis product obtained in step S2 is mixed with a complex bacterium agent for fermentation. After fermentation, it is sterilized and filtered to obtain a fermentation product. S4 Ionic liquid extraction: The fermentation product prepared in step S3 is added to a mixed solution of ionic liquid - ethanol, and CO2 is slowly introduced. Oscillation promotes the extraction reaction. After stopping the introduction of CO2, it is allowed to stand for layer separation and liquid separation. The water layer is removed, dried and then filtered to obtain an extract. S5 Organic solvent extraction: The extract obtained in step S4 is refluxed and extracted with petroleum ether. After filtration, the filtrate is concentrated by rotary evaporation. Subsequently, it is extracted by condensation and reflux with absolute ethanol, filtered by suction and spun dry, and then the organic solvent is removed by vacuum drying to finally obtain the essential oil of Cinnamomum burmannii chvar. Borneol leaves.

[0007] Preferably, the complex enzyme in step S2 is composed of xylanase, hemicellulase, lipase and pectinase, and the enzyme activity ratio is 1.5:2:0.8:1.

[0008] More preferably, the complex bacteria in step S3 are selected from any one of the following groups: Group A: Aspergillus niger + Pichia pastoris + Bacillus amyloliquefaciens; Group B: Trichoderma reesei + Pichia pastoris + Lactobacillus plantarum; Group C: Aspergillus oryzae + Pichia pastoris + Bacillus licheniformis; Group D: Aspergillus terreus + Pichia pastoris + Bacillus subtilis.

[0009] The present invention also provides a complex enzyme, which comprises xylanase, hemicellulase, lipase and pectinase, and the enzyme activity ratio is 1.5:2:0.8:1.

[0010] In some embodiments, the present invention also provides a complex bacterium agent, which is selected from any one of the following groups: Group A: Aspergillus niger + Pichia pastoris + Bacillus amyloliquefaciens; Group B: Trichoderma reesei + Pichia pastoris + Lactobacillus plantarum; Group C: Aspergillus oryzae + Pichia pastoris + Bacillus licheniformis; Group D: Aspergillus terreus + Pichia pastoris + Bacillus subtilis.

[0011] Finally, the present invention provides an application of *Cinnamomum burmannii* var. *camphora*, which is prepared by the above method and is used for preparing a drug or a health product for preventing or treating chronic pharyngitis. By significantly reducing the release of inflammatory mediators such as IFN-β and IL-6, it can alleviate the inflammatory response and inhibit the inflammatory process, thereby achieving the effect of treating or preventing chronic pharyngitis; In some embodiments, the *Cinnamomum burmannii* var. *camphora* simultaneously has the effect of improving local nerve conduction.

[0012] In some embodiments, the *Cinnamomum burmannii* var. *camphora* has a high yield, and at the same time, maximally retains the pharmacological properties of its active ingredients, ensuring its anti-inflammatory and anti-inflammatory effect.

[0013] Compared with the prior art, the present invention has at least the following beneficial effects: First of all, the present invention combines the composite enzyme hydrolysis and composite bacteria fermentation technologies to efficiently destroy the cell structure of *Cinnamomum burmannii* var. *camphora* leaves, thereby greatly improving the extraction efficiency of borneol essential oil; by using composite bacteria fermentation, not only the extraction rate of the active ingredients of borneol is increased, but also its pharmacological properties are significantly enhanced; Secondly, the fungi and bacteria in the composite bacteria agent have rich enzyme secretion capabilities. For example, fungi such as *Aspergillus niger*, *Trichoderma reesei*, *Aspergillus oryzae* and *Aspergillus terreus* can produce highly active cellulase, xylanase, pectinase and hemicellulase. These enzymes can further destroy the main components of the plant cell wall (cellulose, hemicellulose and pectin), thereby releasing the active ingredients inside the cells (including borneol essential oil); during the fermentation process, the composite bacteria agent can convert some precursor compounds (such as terpenoids, flavonoids and polysaccharides) in the *Cinnamomum burmannii* var. *camphora* leaves into more active secondary metabolites through its metabolic pathway, thereby improving the pharmacological properties of the essential oil; in addition, composite bacteria fermentation modifies the plant metabolites (such as hydroxylation, demethylation, etc.), and converts the macromolecular active ingredients into small molecular metabolites that are more easily absorbed by the human body. For example, *Aspergillus niger* and *Aspergillus oryzae* can promote the depolymerization and recombination of terpenoids during the fermentation process, thereby increasing the yield of borneol essential oil.

[0014] Finally, the present invention introduces an extraction technology of green solvents (such as ionic liquid-ethanol mixed solution), reduces the usage of traditional organic solvents, reduces environmental pollution, and at the same time ensures the safety of the extraction products; the borneol essential oil prepared by the present invention has a significant effect in inhibiting the release of inflammatory mediators and improving sleep and relieving mood, and has broad application prospects. Description of the Drawings

[0015] Figure 1 Influence of different composite bacteria fermentation treatments on the yield of borneol essential oil from *Cinnamomum burmannii* var. *camphora*.

[0016] Figure 2Changes in the main components of Borneol-type Cinnamomum burmannii essential oil after fermentation treatment with different compound bacteria.

[0017] Figure 3 Effects of Borneol-type Cinnamomum burmannii essential oil extracted after fermentation treatment with compound bacteria on IL-6 in rats with chronic pharyngitis.

[0018] Figure 4 Effects of Borneol-type Cinnamomum burmannii essential oil extracted after fermentation treatment with compound bacteria on IFN-β in rats with chronic pharyngitis. Specific implementation manners

[0019] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0020] Example 1. Method for extracting Borneol-type Cinnamomum burmannii leaf essential oil with compound bacteria Group A: Aspergillus niger + Pichia pastoris + Bacillus amyloliquefaciens S1 Pretreatment: Crush the dried leaves of Borneol-type Cinnamomum burmannii (Cinnamomum burmannii chvar. Borneol), and pass through a 60-mesh sieve to obtain Borneol-type Cinnamomum burmannii leaf powder; S2 Enzymolysis: Weigh 25 g of the Borneol-type Cinnamomum burmannii leaf powder prepared in step S1, add 1 g of xylanase at 150 U / g, 1 g of hemicellulase at 200 U / g, 1 g of lipase at 80 U / g, and 1 g of pectinase at 100 U / g powders. Using 0.8 mol / L citric acid-sodium citrate buffer solution as the solvent, adjust the pH to 5.5, enzymolyze at 45 °C for 4 hours, heat to 100 °C to inactivate the enzyme for 5 minutes, filter, and retain the solid part; S3 Fermentation: Add the activated Aspergillus niger, Pichia pastoris and Bacillus amyloliquefaciens to the enzymolysis product in step S2. The inoculation amounts are 1.5%, 1.5%, and 1.5% by volume ratio respectively. Ferment and culture at 30 °C for 24 h, sterilize with ultraviolet light, filter, and obtain the fermentation product; S4 Ionic liquid extraction: Add the fermentation product in step S3 to 20 parts by weight of the mixed solution of ionic liquid-ethanol, introduce CO2 at a rate of 0.5 mL / min for 30 min, shake, stop introducing CO2, obtain the high-extraction ionic liquid-ethanol mixed solution, let it stand for stratification, separate the liquid, remove the water layer, wash, dry, filter, and obtain the extract; S5 Organic solvent extraction: Extract the extract in step S4 with petroleum ether by reflux extraction for 1.5 h. After filtration, concentrate it with a rotary evaporator, then carry out condensation reflux with absolute ethanol at 60 °C for 1 h, filter by suction, and after the filtrate is dried by rotation, vacuum dry it to remove the organic solvent, thereby obtaining the Borneol-type Cinnamomum burmannii leaf essential oil of Group A.

[0021] Group B: Trichoderma reesei + Pichia pastoris + Lactobacillus plantarum S1 Pretreatment: Crush the dried leaves of Cinnamomum burmannii chvar. Borneol, and sieve them through a 60-mesh sieve to obtain the powder of Cinnamomum burmannii chvar. Borneol leaves; S2 Enzymolysis: Weigh 25 g of the Cinnamomum burmannii chvar. Borneol leaf powder prepared in step S1, add 1 g of xylanase at 150 U / g, 1 g of hemicellulase at 200 U / g, 1 g of lipase at 80 U / g, and 1 g of pectinase at 100 U / g powders. Using 0.8 mol / L citric acid - sodium citrate buffer solution as the solvent, adjust the pH to 5.5, enzymolyze at 45 °C for 4 hours, heat to 100 °C to inactivate the enzyme for 5 minutes, filter, and retain the solid part; S3 Fermentation: Add the activated Trichoderma reesei, Pichia pastoris, and Lactobacillus plantarum to the enzymolysis product in step S2. The inoculation amounts are 1.5%, 1.5%, and 1.5% by volume ratio respectively. Ferment and culture at 32 °C for 24 h, sterilize with ultraviolet light, filter to obtain the fermentation product; S4 Ionic liquid extraction: Add the fermentation product in step S3 to 20 parts by weight of the mixed solution of ionic liquid - ethanol, introduce CO2 at a rate of 0.5 mL / min for 30 min, shake, stop introducing CO2 to obtain the high-extraction ionic liquid - ethanol mixed solution, let it stand for layering, separate the layers, remove the water layer, wash, dry, filter to obtain the extract; S5 Organic solvent extraction: Reflux and extract the extract in step S4 with petroleum ether for 1.5 h. After filtration, concentrate it with a rotary evaporator, then perform condensation reflux with absolute ethanol at 60 °C for 1 h, filter by suction. After the filtrate is spun dry, dry it under vacuum to remove the organic solvent, thereby obtaining the essential oil of Cinnamomum burmannii chvar. Borneol in group B.

[0022] Group C: Aspergillus oryzae + Pichia pastoris + Bacillus licheniformis S1 Pretreatment: Crush the dried leaves of Cinnamomum burmannii chvar. Borneol, and sieve them through a 60-mesh sieve to obtain the powder of Cinnamomum burmannii chvar. Borneol leaves; S2 Enzymolysis: Weigh 25 g of the Cinnamomum burmannii chvar. Borneol leaf powder prepared in step S1, add 1 g of xylanase at 150 U / g, 1 g of hemicellulase at 200 U / g, 1 g of lipase at 80 U / g, and 1 g of pectinase at 100 U / g powders. Using 0.8 mol / L citric acid - sodium citrate buffer solution as the solvent, adjust the pH to 5.5, enzymolyze at 45 °C for 4 hours, heat to 100 °C to inactivate the enzyme for 5 minutes, filter, and retain the solid part; S3 Fermentation: Add the activated Aspergillus oryzae, Pichia pastoris, and Bacillus licheniformis to the enzymatic hydrolysate obtained in step S2. The inoculation amounts are 1.5%, 1.5%, and 1.5% by volume ratio respectively. Ferment and culture at 30 °C for 24 h, sterilize with ultraviolet light, and filter to obtain the fermentation product; S4 Ionic liquid extraction: Add the fermentation product from step S3 to 20 parts by weight of a mixed solution of ionic liquid - ethanol, introduce CO2 at a rate of 0.5 mL / min for 30 min, shake, stop introducing CO2 to obtain a highly extracted ionic liquid - ethanol mixed solution, let it stand for layering, separate the layers, remove the aqueous layer, wash, dry, filter to obtain the extract; S5 Organic solvent extraction: Reflux and extract the extract from step S4 with petroleum ether for 1.5 h. After filtration, concentrate it with a rotary evaporator, then carry out condensation reflux with absolute ethanol at 60 °C for 1 h, perform suction filtration, spin - dry the filtrate and then dry it under vacuum to remove the organic solvent, thereby obtaining the essential oil of Cinnamomum burmannii var. borneol leaves in group C.

[0023] Group D: Aspergillus terreus + Pichia pastoris + Bacillus subtilis S1 Pretreatment: Crush the dried leaves of Cinnamomum burmannii var. borneol, pass through a 60 - mesh sieve to obtain the powder of Cinnamomum burmannii var. borneol leaves; S2 Enzymolysis: Weigh 25 g of the powder of Cinnamomum burmannii var. borneol leaves prepared in step S1, add 1 g of xylanase (150 U / g), 1 g of hemicellulase (200 U / g), 1 g of lipase (80 U / g), and 1 g of pectinase (100 U / g) powders. Use 0.8 mol / L citric acid - sodium citrate buffer solution as the solvent, adjust the pH to 5.5, carry out enzymatic hydrolysis at 45 °C for 4 h, heat to 100 °C to inactivate the enzyme for 5 min, filter, and retain the solid part; S3 Fermentation: Add the activated Aspergillus terreus, Pichia pastoris, and Bacillus subtilis to the enzymatic hydrolysate obtained in step S2. The inoculation amounts are 1.5%, 1.5%, and 1.5% by volume ratio respectively. Ferment and culture at 32 °C for 24 h, sterilize with ultraviolet light, and filter to obtain the fermentation product; S4 Ionic liquid extraction: Add the fermentation product from step S3 to 20 parts by weight of a mixed solution of ionic liquid - ethanol, introduce CO2 at a rate of 0.5 mL / min for 30 min, shake, stop introducing CO2 to obtain a highly extracted ionic liquid - ethanol mixed solution, let it stand for layering, separate the layers, remove the aqueous layer, wash, dry, filter to obtain the extract; S5 Organic solvent extraction: The extract obtained in step S4 is refluxed and extracted with petroleum ether for 1.5 h. After filtration, it is concentrated by a rotary evaporator, followed by condensation and reflux with absolute ethanol at 60 °C for 1 h. Then, it is suction-filtered. After the filtrate is spun dry, it is vacuum-dried to remove the organic solvent, thereby obtaining the essential oil of the Borneol-type Cinnamomum burmannii leaves in group D.

[0024] Group E: The enzymatic hydrolysate was not fermented with the complex bacteria.

[0025] S1 Pretreatment: The dried Borneol-type Cinnamomum burmannii leaves are crushed and passed through a 60-mesh sieve to obtain the Borneol-type Cinnamomum burmannii leaf powder. S2 Enzymatic hydrolysis: Weigh 25 g of the Borneol-type Cinnamomum burmannii leaf powder prepared in step S1, and add 1 g of xylanase (150 U / g), 1 g of hemicellulase (200 U / g), 1 g of lipase (80 U / g), and 1 g of pectinase (100 U / g) powders. Using 0.8 mol / L citric acid-sodium citrate buffer solution as the solvent, adjust the pH to 5.5, and perform enzymatic hydrolysis at 45 °C for 4 hours. Then, heat to 100 °C to inactivate the enzyme for 5 minutes, filter, and retain the solid part. S3 Ionic liquid extraction: Add the enzymatic hydrolysate obtained in step S2 to a 20-weight part mixed solution of ionic liquid-ethanol, introduce CO2 at a rate of 0.5 mL / min for 30 min, shake, stop introducing CO2, to obtain a high-extraction ionic liquid-ethanol mixed solution. Let it stand for layer separation, separate the layers, remove the aqueous layer, wash, dry, filter, to obtain the extract. S4 Organic solvent extraction: The extract obtained in step S3 is refluxed and extracted with petroleum ether for 1.5 h. After filtration, it is concentrated by a rotary evaporator, followed by condensation and reflux with absolute ethanol at 60 °C for 1 h. Then, it is suction-filtered. After the filtrate is spun dry, it is vacuum-dried to remove the organic solvent, thereby obtaining the essential oil of the Borneol-type Cinnamomum burmannii leaves in group E.

[0026] Yield of essential oil (%) = mass of essential oil obtained by vacuum drying / mass of Borneol-type Cinnamomum burmannii raw material × 100% The essential oil chemical components were detected by gas chromatography-mass spectrometry (GC-MS). The instrument used was a Shimadzu GCMS-QP2020W / O gas chromatography-mass spectrometer from Japan; the gas chromatography conditions were as follows: SH-RxiTM-5Sil MS chromatographic column (30m×0.25 mm, 0.25μm); temperature programming: the initial column temperature was 70°C, heated to 160°C at a rate of 2°C / min, held for 2 min, then heated to 220°C at a rate of 10°C / min, held for 5 min, and the whole process ran for 51 min. The injection volume was 0.5 μL, split injection was used, and the injection port temperature was 230°C; the carrier gas was nitrogen, and the constant flow rate of the carrier gas through the column was 1.19 mL / min. Mass spectrometry conditions (MS): ion source temperature 200°C; scanning mass range m / z50~500, to statistically analyze the effects of different complex bacteria treatments on the yield of Borneol-type Cinnamomum burmannii essential oil, see Figure 1 ; at the same time, the main components of the essential oils in groups A-E were analyzed using the GC-MS instrument and parameters, and the results are shown in Figure 2 .

[0027] Figure 1 The results showed that there were significant differences in the effects of different complex bacteria fermentation treatments in groups A-D on the yield of Borneol-type Cinnamomum burmannii essential oil compared with the treatment in group E. Among them, the difference in the yield of Borneol-type Cinnamomum burmannii essential oil by the complex bacteria fermentation treatment in group B was the most significant (p < 0.01), and the effect of the complex bacteria fermentation treatment in group B on the yield of Borneol-type Cinnamomum burmannii essential oil was significantly higher than those in the other groups A, C, and D (p < 0.05), and the essential oil yield was the highest.

[0028] Figure 2 The results showed that the release of effective compounds including Borneol, 1,8-Cineole, Camphor, and α-Pinene in the leaves of Borneol-type Cinnamomum burmannii by the complex bacteria fermentation treatment in groups A-D was significantly increased compared with that in group E, especially for Borneol (p < 0.01) and 1,8-Cineole (p < 0.05); among them, the complex bacteria fermentation treatment in group B could more significantly promote the release of effective compounds including Borneol, 1,8-Cineole, Camphor, and α-Pinene compared with the complex bacteria fermentation treatments in groups A, C, and D.

[0029] Example 2. Study on the effect of complex bacteria fermentation-assisted extraction of essential oil from Borneol-type Cinnamomum burmannii leaves on a chronic pharyngitis mouse model Forty-eight male SD rats were randomly divided into a blank group, a model group, Group A, Group B, Group C, Group D, Group E, and a throat-clearing group, with a total of 8 groups, 8 rats in each group. They were marked with picric acid, and the weight of each rat was recorded. The rats in each group were fed with ordinary feed and given free access to water. In the model group, Group A, Group B, Group C, Group D, Group E, and the throat-clearing group, the pharynx of the animals was sprayed with 2.5% ammonia water once in the morning and afternoon from the 1st to the 15th day, and 3 presses were sprayed each time with a laryngeal nebulizer; in the blank group, distilled water was sprayed on the pharynx in the same way from the 1st to the 15th day. The food intake, activity level, water intake, oral secretions, and whether there were actions of scratching the oropharynx of the rats were observed daily, and the pharynx of the rats was observed once a day. After determining that the model was successfully established, from the 16th to the 25th day, the mice in the throat-clearing group were administered the Chinese patent medicine Houyanqing Granules (composed of Achyranthes bidentata Blume, Kalimeris indica (L.) Sch.-Bip., Plantago asiatica L., and Carpesium abrotanoides L.) at a dose of 10 g / kg per day. When used, it was prepared into a solution with a concentration of 0.1 g / ml with 55°C distilled water for pharyngeal spraying; in Group A, Group B, Group C, Group D, and Group E, the essential oil of Cinnamomum burmanni (Nees) Blume leaves of the Borneol type prepared in Groups A-E of Example 1 was administered at a dose of 1 g / kg per day. When used, it was prepared into a solution with a concentration of 0.1 g / ml with 55°C distilled water for pharyngeal spraying; when used, it was prepared into a solution with the same volume as that of the throat-clearing group with 55°C distilled water for pharyngeal spraying; the blank group and the model group were given the same volume of distilled water. Finally, ELISA was used to detect IL-6 and IFN-β, see Figure 3 。

[0030] Figure 3 The results showed that compared with the blank group, the IL-6 level in the model group was significantly increased (P < 0.01), indicating that the chronic pharyngitis mouse model was successfully established; compared with the model group, the IL-6 level in the positive control throat-clearing group was significantly decreased, and the essential oil of Cinnamomum burmanni (Nees) Blume leaves of the Borneol type in Group B had a significant effect of reducing the secretion of serum IL-6 compared with the essential oils of Cinnamomum burmanni (Nees) Blume leaves of the Borneol type in Groups A, C, and D (P < 0.01).

[0031] Figure 4 The results showed that compared with the blank group, the IFN-β level in the model group was significantly increased (P < 0.05), indicating that the chronic pharyngitis mouse model was successfully established; compared with the model group, the IFN-β level in the positive control throat-clearing group was significantly decreased, and the essential oil of Cinnamomum burmanni (Nees) Blume leaves of the Borneol type in Group B had a significant effect of reducing the secretion of serum IFN-β compared with the essential oils of Cinnamomum burmanni (Nees) Blume leaves of the Borneol type in Groups A, C, and D (P < 0.01).

[0032] Test on the mood soothing effect of the essential oil of Cinnamomum burmanni (Nees) Blume leaves of the Borneol type assisted by compound bacteria fermentation in Example 3 This study adopted a randomized controlled trial design, and eligible volunteers were randomly divided into groups A - E. When recruiting volunteers, all participants signed up voluntarily and were confirmed to have a certain tendency of sleep disorder during the initial screening process. To ensure the scientificity and reliability of the experiment, all volunteers needed to fill out the PSQI scale before the study to record their subjective sleep quality and other relevant health data.

[0033] At the beginning of the experiment, the volunteers would receive a 30 - day treatment with essential oils. During this period, the experimental group used the essential oils fermented by the composite bacteria in groups A - D and the essential oil in group E for aromatherapy or topical application every day. All participants needed to perform the same bedtime essential oil application procedure every evening. At the same time, the volunteers needed to continue filling out the PSQI scale and subjective sleep quality evaluations to record the improvement of sleep and related emotional changes.

[0034] The Pittsburgh Sleep Quality Index (PSQI) scale was used for subjective sleep quality evaluation: It consists of 19 self - rating and 5 other - rating items. The 19th self - rating item and the 5th other - rating item are not scored. The remaining 18 scored items are divided into 7 dimensions: sleep quality, sleep latency, sleep duration, sleep disturbances, sleep medications, and daytime dysfunction. Each dimension is scored from 0 - 3, and the cumulative scores of each dimension are the total PSQI score. The score ranges from 0 - 21 points. A score > 7 points indicates poor sleep quality, and a score ≤ 7 points indicates good sleep quality. When the score drops by ≥ 3 points before and after the test, it is considered that the subjective sleep has improved.

[0035] Table 1 Emotional soothing effect test of the essential oil from the leaves of Cinnamomum burmanni var. austrosinense with borneol

[0036] It can be seen from the test results that the essential oil from the leaves of Cinnamomum burmanni var. austrosinense assisted by composite bacteria fermentation in the present invention has a certain effect on improving sleep. The scores before and after the test both dropped by ≥ 3 points, and the sleep improvement effect is good, and it has a certain emotional soothing effect.

[0037] Example 4: The essential oil improves the scalp environment Volunteer test: 25 middle - aged men and 25 middle - aged women were selected, with dry hair quality or excessive hair oiliness, and they used the essential oil of Example 1 once a day. Specific usage method: Apply 2 mL of the essential oil in group A of Example 1 on the comb or the hair roots on the top of the head, comb along the hair for 5 minutes, and then massage the hair roots with hands for 5 minutes. After 15 days, score the used hair care essential oil on a 10 - point scale, where a higher score represents better performance; scoring rules: 1 - 2 points is "very dissatisfied", 3 - 4 points is "dissatisfied", 5 - 6 points is "average", 7 - 8 points is "satisfied", and 9 - 10 points is "very satisfied".

[0038] By calculating the proportion of the number of people who give positive feedback (selecting "very satisfied" and "satisfied") to the total number of people giving evaluations, the degree of recognition of the subjects for the overall effect can be understood. When the satisfaction rate is greater than 50%, it indicates that the product has the corresponding efficacy.

[0039] The statistical results show that: 20% of the subjects (10 people) scored between 9 and 10 points and were very satisfied; 70% of the subjects (35 people) scored between 7 and 8 points and were satisfied; the above-mentioned subjects (45 people) accounted for 90% of the total number of surveyed people, and among these 90% of the subjects, 80% of the subjects (36 people) thought that the essential oil of the present invention could effectively clean the scalp oil, reduce dandruff, and reduce the problem of dry hair; 10% of the subjects (5 people) scored between 5 and 6 points and thought it was average. Generally speaking, it shows that the essential oil of the present invention can improve the scalp environment and is used for head massage and hair care.

[0040] The above is the preferred implementation manner of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for extracting borneol by compound bacteria fermentation, characterized in that It includes the following steps: S1 Pretreatment: The leaves of Cinnamomum burmannii chvar. Borneol are mixed and pulverized after microwave drying. S2 Enzymolysis: The solid obtained in step S1 is added to a citric acid-sodium citrate buffer solution, and a complex enzyme is added for enzymolysis. The enzyme is inactivated to obtain an enzymolysis product, which is filtered under reduced pressure and vacuum dried. S3 Fermentation: The enzymolysis product in step S2 is added with a complex bacterium for fermentation culture, sterilized, and filtered to obtain a fermentation product. S4 Ionic liquid extraction: The fermentation product prepared in step S4 is added to a mixed solution of ionic liquid-ethanol, CO2 is slowly introduced, shaken, and then the introduction of CO2 is stopped to obtain a high-extraction ionic liquid-ethanol mixed solution. It is allowed to stand for stratification, separated, the water layer is removed, dried, filtered to obtain an extract. S5 Organic solvent extraction: The extract in step S4 is refluxed and extracted with petroleum ether, filtered, the filtrate is concentrated by a rotary evaporator, vacuum dried, condensed and refluxed with absolute ethanol, filtered by suction, the filtrate is spun dry and then vacuum dried to remove the organic solvent to obtain the essential oil of Cinnamomum burmannii chvar. Borneol leaves.

2. The method according to claim 1, characterized in that The complex enzyme in step S2 is composed of xylanase, hemicellulase, lipase and pectinase, and the enzyme activity ratio is 1.5:2:0.8:

1.

3. The method according to claim 1 or 2, characterized in that, The complex bacterium in step S3 is selected from any one of the following groups: Group A: Aspergillus niger + Pichia pastoris + Bacillus amyloliquefaciens; Group B: Trichoderma reesei + Pichia pastoris + Lactobacillus plantarum; Group C: Aspergillus oryzae + Pichia pastoris + Bacillus licheniformis; Group D: Aspergillus terreus + Pichia pastoris + Bacillus subtilis.

4. Application of a composite bacterium in increasing the yield of Cinnamomum burmanni var. austrosinense Hance with borneol, characterized in that, The complex bacterium is selected from any one of the following groups: Group A: Aspergillus niger + Pichia pastoris + Bacillus amyloliquefaciens; Group B: Trichoderma reesei + Pichia pastoris + Lactobacillus plantarum; Group C: Aspergillus oryzae + Pichia pastoris + Bacillus licheniformis; Group D: Aspergillus terreus + Pichia pastoris + Bacillus subtilis.

5. Use of the borneol prepared by the method according to any one of claims 1-3 in the preparation of a product for preventing or treating chronic pharyngitis.

6. The application according to claim 5, wherein The product is a health product or a drug.

7. Use of the borneol prepared by the method according to any one of claims 1-3 in the preparation of a product for improving sleep and relieving mood.

8. The application according to claim 7, wherein The product is a cosmetic.

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